Spats 1 (Srsp1) is differentially expressed during testis development of the rat.

Spats1 encodes the first reported testis-specific protein containing a long serine stretch. Besides, it bears a probable bipartite nuclear localization signal. Here, we describe the expression pattern of Spats1 in rat along embryonic and postnatal testis development by immunoblots and confocal immunohistochemistry. Spats1 is first expressed in the embryo at 17.5 days post-coitum, coinciding with the time when gonocytes acquire a quiescent state. At this time expression is detected in peritubular myoid cells and gonocytes. Spats1 attains maximum levels during meiosis of the first spermatogenic wave, mainly in pachytene spermatocytes, while a lower signal is also observed in spermatogonia, Sertoli cells and myoid cells. Protein levels dramatically decay afterwards, with minimum expression in adult individuals, where no signal was detected in elongating spermatids or spermatozoa. Spats1 is mostly cytoplasmic, although in pachytene spermatocytes it mapped to nuclei as well. Alkaline phosphatase treatment showed that this protein would be highly phosphorylated. Moreover, we show that the protein is highly conserved along metazoan evolution. Our results suggest a role in the initiation of the first spermatogenic wave, and in the establishment or progression of the first male meiotic division.

Ultra-fast and optimized method for the preparation of rodent testicular cells for flow cytometric analysis.

UNLABELLED: Homogeneity of cell populations is a prerequisite for the analysis of biochemical and molecular events during male gamete differentiation. Given the complex organization of the mammalian testicular tissue, various methods have been used to obtain enriched or purified cell populations, including flow cell sorting. Current protocols are usually time-consuming and may imply loss of short-lived RNAs, which is undesirable for expression profiling. We describe an optimized method to speed up the preparation of suitable testicular cell suspensions for cytometric analysis of different spermatogenic stages from rodents. The procedure takes only 15 min including testis dissection, tissue cutting, and processing through the Medimachine System (Becton Dickinson). This method could be a substitute for the more tedious and time-consuming cell preparation techniques currently in use. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (DOI:10.1007/s12575-009-9003-2) contains supplementary material, which is available to authorized users.

Molecular cloning and analysis of a DNA repetitive element from the mouse genome.

We report the development of a 3-week laboratory activity for an undergraduate molecular biology course. This activity introduces students to the practice of basic molecular techniques such as restriction enzyme digestion, agarose gel electrophoresis, cloning, plasmid DNA purification, Southern blotting, and sequencing. Students learn how to carry out a GenBank™ search as they are encouraged to compile most available information about their cloned sequence. The natural integration of the bench work with the use of the bioinformatics tools is considered a major advantage of this laboratory course.

The mammalian gene pecanex 1 is differentially expressed during spermatogenesis.

Using mRNA differential display and cDNA library screening approaches we have identified differential gene expression of pecanex 1--a mammalian homologue of pecanex gene from Drosophila--in the testes of the rat. Northern blot analyses showed that the transcript is only present in the germ line and not in the somatic cells of the testis, reaching its peak at the pachytene stage of the meiotic prophase. Moreover, nonradioactive in situ hybridization did not detect the expression of the gene in any cell type of the testis other than pachytene spermatocytes. Northern blot assays did not allow the detection of the transcript in nine other tissues. Remarkably, although pecanex exerts a neurogenic role in Drosophila, the transcript was not detectable by Northern blotting in the nervous tissue of adult rats, nor in the brain of neonate and embryonal stages. The protein product of the pecanex 1 gene was detected by immunoblotting in pachytene spermatocytes and round spermatids as well, but not in liver nor brain. From genomic analysis we conclude that, although only one pecanex gene exists in Drosophila, mammalian pecanex 1 belongs to a gene family with three related genes in different chromosomes. We speculate that pecanex 1 could play an important role in the testis, related to spermatogenesis.

Quantitative study on guinea pig spermatogenesis shows a relative high percentage of early meiotic prophase stages.

Meiosis is the special double cellular division characterized by the reduction of chromosome number of the final products and recombination of genetic information present in maternal and paternal homologous chromosomes. Early stages of meiotic prophase, leptotene and zygotene (L/Z), are functionally important since homologous chromosomes recognize, align, and pair during them. They are poorly represented in the seminiferous tubules of mammalian species, and this fact turns studies focused on these stages difficult to perform. As a consequence, the molecular bases of these important events are so far poorly known and understood in higher eukaryotes. The purpose of this work was to provide an advantageous experimental mammalian model (with a reasonable number of cells) for biochemical and molecular analysis of early meiotic prophase stages. Here, we present the results of our quantitative study on testes material of both immature and adult guinea pig specimens (Cavia porcellus). We show that their seminiferous tubules contain a comparatively high percentage of L/Z spermatocytes, as well as a very conspicuous chromosome bouquet at the L/Z transition, which points out this species as a well-suited one to address studies on such stages in mammals.

Down-regulation of photosystem I by Erwinia carotovora-derived elicitors correlates with H(2)O(2) accumulation in chloroplasts of potato.

SUMMARY Identification of Solanum tuberosum genes responsive to culture filtrates (CF) from Erwinia carotovora ssp. carotovora resulted in isolation of psaD, a nuclear gene encoding the PSI-D subunit of photosystem I (PSI). This gene was rapidly and markedly down-regulated in CF-treated or wounded plants. Down-regulation of psaD transcripts was also triggered by signal molecules involved in plant defence such as methyl jasmonate. The CF-induced down-regulation of psaD transcripts was correlated with an accumulation of hydrogen peroxide in chloroplasts and a down-regulation of the NADP(+) photoreduction activity mediated by PSI. These results suggest that the CF-induced down-regulation of PSI may be related to the accumulation of reactive oxygen species in chloroplasts of plant cells responding to E. c. carotovora.

A novel potato defence-related alcohol:NADP+ oxidoreductase induced in response to Erwinia carotovora.

Identification of Solanum tuberosum genes responsive to culture filtrates (CF) from Erwinia carotovora subsp. carotovora led to the isolation of a full-length cDNA with high sequence similarity to several alcohol dehydrogenases. Accumulation of transcripts corresponding to this defence-related alcohol dehydrogenase (drd-1) was rapidly induced in CF-treated and wounded plants. The gene was also responsive to molecules involved in defence signalling such as salicylic acid, methyl jasmonate and ethylene. To elucidate the biochemical function of DRD-1, its cDNA was expressed in Escherichia coli. Enzymatic assays revealed that DRD-1 is an alcohol:NADP+ oxidoreductase with preference for various aromatic and aliphatic aldehydes. The enzyme exhibited high activity with several aldehydes including 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, salicylaldehyde, o-vanillin, cinnamaldehyde, hydrocinnamaldehyde, hexanal and octanal. Identification of the reaction product by thin-layer chromatography confirmed the reduction of aldehydes to alcohols. Enzymatic activity measured with 2-methoxybenzaldehyde as a substrate was increased in salicylic acid- or methyl jasmonate-treated plants. These data suggest that DRD-1 may play an important role in potato defence response to Erwinia carotovora.